Derivatives of 7-aminocephalosporanic acid



United States Patent 3,173,916 DERIVATIVES or marvmvocnrnarosronamc ACIDGilbert M. Shall, Old Lyme, and Ben A. Schin, Gales Ferry, (101111.,assignors to Chas. Pfizer & Co., Inc New York, N .Y., a corporation ofDelaware No Drawing. Filed June 28, 1960, Ser. No. 39,218 15 Claims.(Cl; 260-243) This invention relates to new antibiotic compounds andtheir salts which are active against both gram-positive andgram-negative microorganisms and resistant to penicillinase.

Cephalosporin C, a valuable antibiotic having the Formula IA wherein Rrepresents the D-4-arnino-4-carboXy-nbuty1 moiety is producedsimultaneously with Cephalosporin N, a pencillin type compound alsoknown as Synnernatin B and (D-4-anrino-4-carboxy-n-butyl)-penicillin, bya species of Cephalosporium. t resembles Cephalosporin N in many of itsphysical, biological and chemical properties. In many respectsCephalosporiu C resembles the peniciilins in general in that it is-anN-acyl derivative of a fi-lactam fused to a dihydrothiazine ring systemin place of a fused B-lactamthiazolidine ring system. It reacts withweak heterocyclic tertiary bases, such as pyridine, to form a compoundbelieved to have the Formula 13 which exists as the Zwitter ion form andis known trivially as Cephalosporin C The Nacyl side chain can, byanalogy to amide linkages in general and to the amide linkage ofpencillins in particular, be cleaved by mild hydrolysis to produce theprecursor compound. Hydrolysis of the acyl moiety in the penicillinseries, for example, produces the penicillin nucleus or precursorG-aminopenicillanio, acid. Similarly, hydrolysis of the acyl group ofCephalosporin C produces the corresponding nucleus or precursor'I-amitogether with the lactone of the desacetyl derivative ofCephalosporin C, known as Cephalosporin C Further hydrolysis ofCephalosporin C removes the D--aminofi-carboxyvaleryl side chain to givethe lactone of the ice desacetyl derivative of 7-aminocephalosporanicacid having Formula III:

in the same manner, Cephalosporin C is hydrolyzed to its correspondingnucleus or precursor having Formula HB. As expected, the properties ofCephalosporin C, particularly the antibiotic properties, are dependentupon the overall structure of the compound. However, as in thepenicillin seriesof compounds it is expected that the properties of aparticular cephalosporin, as the N-acyl derivatives of7-aminocephalosporanic acid will hereinafter he referred to, willdependupon the acyl group attached'to the amino group of theprecursorcompounds of Formulae HA and HB.

The novel antibiotics of the present invention'will be named forconvenience by reference to the particular R group attached to the -CONHgroup of the cephalosporanic acid nucleus. Thus, for example, thecompound of Formula IA wherein R represents theD-4-amino-4-carboxy-n-butyl radical, known trivially asCephalosporin C,is named (D-4-amino-4-carboxy-n-butyl)-cephalosphorin; the compoundwherein'R is the benzyl radical is named benzylcephalosporin. Thecephalosporins derived from Formula 1B which, when R is theD-4-a1nino-4-carboXy-n-butyl, group is known as Cephalosporin C willhereinafter be referred to as the Cephalosporin- A series. Thus, when Ris benzyl, 11B is named benzyl- .cephalos-porin-A. In like manner, theCephalosporins derived from Formula I11 will be referred to as theCephalosporin lactones.

Of the many penicillins known, many are of poor acid stability and onlya few are antagonistic toward both gram-positive and gram-negativemicroorganisms. Fewer still are resistant to peniciilinase.Cephalosporin C, however, shows activity against both gram-positive andgramnegative microorganisms, is stable in the acid pH, range andresistant to penicillinase. Despite theadvantages of Cephalosporin Cover the peniciilins-in general, however, the development'of antibioticshaving increased overall activity, or perhaps increased gram-negativeactivity, relative to that of Cephalosporin C is highly desirable.

There has now been discovered a series of novel and valuablecephalosporins, or N-acyl derivatives of cephalosporanic acid, whichexhibit significant activity toward gram-negative and gram positiveorganisms and the penicillinase producing strains of staphylococci. Thenovel compounds of this invention have, in the acid form, the formulaeillustrated by IA and 1B above wherein R is selected from the groupconsisting of 0cand fl-halo substituted alkyl containing from 1' to 6carbon atoms; a-phenyl-Bhydroxyethyl and tit-substitutedphenyl-fl-hydroxyethyi wherein the substituent is selected from thegroup consisting of alltyl containing 1 to 4 carbon atoms, halogen,trifiuoromethyl, alkoxy containing 1 to 4 carbon atoms, nitro, amino andhydroxyl; amino and substituted amino wherein the substituent isselected from the group consisting of alkyl containing 1 to 10 carbonatoms, phenyl, p-dimethylaminophenyl, nitrophenyl, p-carbomethoxyphenyl,p-chlorophenyl, p-methoxyphenyl and naphthyl; amino alkyl wherein thealkyl group is selected from the group consisting of phenyl substitutedethylene, and straight chain and branched chain alkylene radicalscontaining up to 8 carbon atoms and the N-carbobenzoxy derivativesthereof; a-allylmercaptoalkyl wherein alkyl is selected from the groupconsisting of alkyl containing 1 to 6 carbon atoms; cis: andtrans-styrylmercaptomethyl, n-pentyl, 2-pentenyl, n-heptyl,phenylmercaptomethyl, phydroxybenzyl, a-phenylmercapto-B-hydroxyalkyland ccphenoxy-fl-hydroxyalkyl wherein the alkyl group contains from 2 tocarbon atoms, and the fl-acyl derivatives thereof wherein the acyl groupis selected from the group consisting of the acyl radicals of ahydrocarbon monocarboxylic acid containing 1 to 4 carbon atoms;

wherein M is selected from the group consisting of hydroxy, alkoxycontaining 1 to 4 carbon atoms, amido, carboxyrnethylimido, andcarboxyethylideneimido; R is selected from the group consisting ofmethylene and ethylidene; R is selected from the group consisting ofcycloalkylene containing 4 to 6 carbon atoms, phenylene, 2,5-thienylene,2-carboxy-3,4-thienylene, methylene and substituted methylene whereinthe substituent is selected from the group consisting of carboxy, andhalogen; alkylene containing 2 to 4 carbon atoms which may beunsaturated and substituted derivatives thereof wherein the substituentis selected from the group consisting of hydroxy, carboxy, halogen andalkanoyloxy radical of a hydrocarbon carboxylic acid containing 1 to 4carbon atoms; and R is selected from the group consisting of acetoxy andl-pyridyl.

- Included also in the present invention are the prod ucts derived fromdesacetyl-7-aminocephalosporanic acid lactone (III), and thephysiologically acceptable salts of these products such as the sodium,potassium, calcium, ammonium and amine salts, all of which possesssignificant antibacterial activity.

The valuable products of this invention are remarkably efiective intreating a number of susceptible grampositive and gram-negativeinfections in animals including man. For this purpose, the purematerials or mixtures thereof with other antibiotics can be employed.They may be administered alone or in combination with a pharmaceuticalcarrier selected on the basis of the chosen route of administration andstandard pharmaceutical practice. For example, they may be administeredorally in the form of tablets containing such excipients as starch, milksugar, certain types of clay, etc., or in capsules alone or in admixturewith the same or equivalent excipients. They may also .be administeredorally in the form of elixirs or oral suspensions which may containflavoring or coloring agents, or be injected parenterally, that is, forexample, intramuscularly or subcutaneously. For parenteraladministration they are best used in the form of a sterile aqueoussolution which may contain other solutes, for example, enough saline orglucose to make the solution isotonic.

The novel and valuable products of this invention are prepared byacylation of the appropriate precursor compound (IIA, 1113 or III) witha slight excess of the proper acid chloride, bromide or anhydride. Theacid anhydrides, preferably the simple anhydrides, are advantageouslyused as acylating agents since their use not only simplifies isolationof the desired product but also avoids the presence of the stronganions, chloride and bromide, which accelerate or cause decomposition ofthe cephalosporin during isolation. However, when an acid halide isemployed as acylating agent the strong anion, if desired, is removed byadsorption on a strong anion exchange resin. The acylat-ion is conductedat a pH of from about 6.0 to about 8.0 and preferably at pH 7.0. Thereaction system is buttered at the desired pH with, for example sodiumbicarbonate, or dilute alkali is added as required throughout thereaction to maintain the desired pH level. The temperature may rangefrom about 0 C. to about 50 C. but is preferably held at from about 0 C.to about 30 C.

The necessary acid chlorides, the side chain precursors, are preparedfor the most part by methods known in the art. Theu-phenyl-e-hydroxyalkanoic acids, for example, are prepared from theappropriately substituted acetophenones according to the proceduredescribed by McKenzie et al., J. Chem. Soc 115, 830 (1919), wh chcomprises: reacting the proper acetophenone derivat ve with hydrogencyanide to produce the corresponding acetophenone cyanohydrin;hydrolyzing the cyanohydrm to the corresponding atrolactinic acid,followed by dehydration of the atrolactinic acid to the correspond ngatropic acid. Treatment of the atropic acid derivative with hydrogenchloride produces the OL-SllbStltlltfid phenylfi-chloropropionic acidwhich is then hydrolyzed to the desired tat-substitutedphenyl-e-hydroxypropionic acid.

Alternatively, the desired OL-SllbStltUtfid phenyl-[B-hydroxypropionicacid is prepared from a suitably substituted pheny-l acetic acid asdescribed by Muller, Ber. 51, 252 (1918), and by Wislicenus et al, Ber.51, 1237 (1918). This method comprises the condensation of a phenylacetic acid ester with a formic ester in the presence of sodium:ethylate followed by reduction of the formyl derivative thus producedto the corresponding hydroxy acid ester. The free acid is obtained bybydrolysis.

The cisand trans-styrylmercaptoacetic acids are prepared via thecondensation of phenylacetylene with thinglycolic acid and the mixtureof cisand trans-forms separated bymeans of their barium salts. Theoc-HllY-lmercaptoalkanoic acids are obtained from the correspondinga-bromoalkanoic acids by reaction with iallyl mercaptan in the presenceof an acid acceptor, or by reaction of the a-mercaptoalkanoic acid withallyl bromide under similar conditions.

The 0,0- disubstituted chlorothionophosphates and phosphates areavailable via the reaction of the appropr-iate alcohol or phenol withthiono phosphoryl chloride or phosphoryl chloride.

1 The a-plrenoxy-fi-hydroxy-alkanoic acids are prepared by theacyl-ation of an ester of phenoxyacetic acid, such as the ethyl ester,with ethylformate in the presence of sodium followed by reduction of thethus produced formyl ester to the fi-hydroxyester. Replacement of ethylformate by esters of alkanoic acids containing up to six carbon atomsproduces the required a-phenoxy-fi-hy droxy alkanoic acids. Theu-phenylmercapto-fi-hydroxyallcanoic acids are prepared in the samemanner from u-phenyl mercaptoacetic acid esters. Acylation of thefl-hydroxy acids or esters with the appropriate acylating agent producesthe corresponding fl-alkanoxyloxy derivatives. i

The required starting acids of formula HOOCR R SR -COOH are obtained bythe condensation of the thio acid,

HOOC-R SH such as thioglycolic acid, with the di-chloro or dibromoderivative of the R moiety, for example methylene bromide, in thepresence of an alkalizing agent, such as. sodium hydroxide or potassiumhydroxide. A modified procedure which is also applicable involves thecondensation of the cuprous salt of the thio acid,

with the dihalo substituted R moiety. This method is useful forpreparing those acids wherein R is phenylene When the two sulfur atomsare bridged by a single carbon atom, i.e., methylene, ethylidene,carboxyrnethylene, the compounds can, in many cases, be prepared fromthe corresponding forrnyl derivative, such as formaldehyde,acetald-ehyde, glyoxylic acid, and the appropriate mercaptan, forexample, thioglycolic acid, according to conventional procedures.

The starting acids thus obtained are converted to the monofunctionalderivatives by conventional methods, such as by reaction of therespective anhydride, prepared by self-condensation of the acid by1,3-dicyclohexylcarbodiimide, with the appropriate M-H reactant, i.e.,methanol, ammonia, glycine. The acid anhydrides, chlorides and bromidesare prepared according to conventional methods.

The acid chlorides of the acids are prepared by reacting the desiredacid with thionyl chloride according to conventional procedures.

When using the precursor acid KB as starting material a buffer systememploying an organic base, such as pyridine-acetate, can be used. Theuse of such a buffer system with 7-cephalosporanic acid or the lactoneof its desacetyl derivat ve brings about their partial transformation tothe Cephalosporin C type compound (18).

Those compounds of this invention wherein R represents the amino orsubstituted amino group as defined above are best prepared bycondensation of '7-arninocephalosporanic acid with the appropriatealkali metal cyanate or isocyanate compound, for example, sodium,potassium or ammonium cyanate, and alkyl and aryl isocyanates.

in the preparation of the aminoalkylcephalosporins, it is desirable, inorder to minimize side reactions and thus obtain optimum yields, toprotect or mask the amino group of the amino acid. The carbobenzoxygroup is the preferred protecting group since it is readily removedunder mild conditions to regenerate the free amino group. Otherprotecting groups, for example, the triphenylmethyl or trityl group, isalso operative in the preparation of these novel penicillins. However,triphenylmethyl chloride is less readily available then carbobenzoxychloride and the trityl group is somewhat more diflicult to remove.

The required precursor compounds (Formulae HA and HE) are obtained bymild acid hydrolysis of Cephalosporin C and Cephalosporin Crespectively. The precursor IIB can also be obtained by treatment of7-aminocephalosporanic acid with pyridine. The precursor com pounds thusproduced possess the same steric structure as the corresponding moietiesof Cephalosporins C and C More specifically, the precursor compounds,7-aminocephalosporanic acid and its desacetyl lactone (IIA and III) areproduced from Cephalosporin C by treating Cephalosporin C with anaqueous solution of a dilute mineral acid, for example hydrochloric acidof from about 0.01 to about 1.0 molarity and a temperature of from aboutto about 50 C. for a period of from several hours to several days. Theperiod of hydrolysis depends, of course, upon the concentration of acidused and the temperature. When using 0.1 M HCl at room temperature, aperiod of 3 to 4 days generally effects substantially completehydrolysis. Alternatively, the hydrolysis can be accomplished by meansof a strong cation exchange resin (H+ cycle). Similarly, hydrolysis ofCephalosporin C produces the precursor 1TB. The products are isolated byelectrophoretic or chromatographic techniques and acylated to thedesired cephalosporin.

The precursor compounds, however, need not be isolated but can beacylated directly to the desired cephalosporin which is then separatedby appropriate methods, for example, chromatographic or electrophoretictechniques.

In addition, the desacetyl-7-aminocephalosporanic acid lactone can bereacted with hydrocarbon carboxylic acids or salts thereof to producethe corresponding hydroxy ester. For example, sodium acetate reacts withthe lactone to regenerate 7-aminocephalosporanic acid. In this manner, avariety of homologs of 7-aminocephalosporanic acid of Formula HA whereinthe R moiety represents an alkanoxyloxy moiety derived from ahydrocarbon carboxylic acid containing up to six or more carbon atomsare prepared. The homologs can in turn be acylated at the 7-amino groupto produce novel isotelic antibiotics having use in the treatment of avariety of bacterial infections in animals and man.

This invention is further illustrated by the following examples, whichare not to be construed as imposing any limitations on the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications and equivalentsthereof which readily suggest themselves to those skilled in the artwithout departing from the spirit of the present invention and/or thescope of the appended claims.

By virtue of the asymmetric center present in the side chain many of thenovel compounds of this invention can exist in diastereoisomeric forms,and mixtures thereof, derived respectively from the isomeric precursoracids. The preparation of the various modifications is most convenientlyand preferably accomplished by reacting the 7-aminocephalosporanicacid'with the appropriate modification, that is, with the dlor the dorthe l-component of the acid chloride.

7-AMINOCEPHALOSPORANIC ACID Procedure A A solution of 1 part (by weight)Cephalosporin C sodium salt in 15 parts (by weight) water is adjusted topH 2.5 by the addition of Dowex 50 x8, hydrogen form (a nuclearsulfonated polystyrene resin crosslinked with divinylbenzene anddescribed in U.S. Patents 2,366,077 and 2,518,420, available from theDow Chemical (30.). The resin is removed by filtration and washed with 5parts (by weight) Water. N-HCl, 5 ml. per part of Cephalosporin C sodiumsalt, is added to the combined filtrate and washings and the solutionkept at room ternperature for 3 days then passed through a column of 2-5ml. of Dowexd, acetate form (a polystyrene product cross linked withdivinylbenzene and containing functional quaternary ammonium groups;available from the Dow Chemical Co.). The column is washed with ml. ofwater followed by 300 ml. of 0.5 N acetic acid. The eluate collectedfrom the water Wash, on concentration in vacuo yields Cephalosporin CThe acetic acid eluate is freeze dried to give 7-aminocephalosporanicacid and other products of the mild acid degradation; notablyCephalosporin C and unchanged Cephalosporin C.

It must be pointed out that when using 7-aminocephalosporanic acid,prepared as described above, in the following examples, theCephalosporin C and Cephalosporin C present are also converted to theirN-acyl derivatives. The product actually obtained is, in each case, amixture of these products in which the aeylated cephalosporinpredominates.

In like manner, Cephalosporin C and Cephalosporin C A are hydrolyzed totheir respective precursors, III and I113. The products are separated byelectrophoresis.

Example I To a solution of 7-aminocephalosporanic acid (prepared asdescribed in Procedure A), neutralized and buffered at pH 7.0 with a 1:2solution of acetone-3% aqueous sodium bicarbonate, there is graduallyadded a 10% excess of fi-brornopropionyl chloride (based on theCephalosporin C used) in acetone. The temperature is maintained at 0-5C. throughout the reaction. After one hour, the mixture is extractedwith an equal volume of ether and the ether layer discarded. The aqueouslayer is adjusted to pH 2.0 with aqueous phosphoric acid and thesolution extracted twice with an equal volume of n-butylacetate. Thecombined n-butyl acetate extracts are washed with volume of water, thenextracted with one-halfvolume of water, sufficient aqueous KOH beingadded to bring the pH to 5.0. The aqueous extract is freeze dried togive a mixture of the potassium salts of [ii-bromoethylcephalosporin,,B-bromoethyleephalosporin C and B-bromoethyl Cephalosporin C. Paperchromatography in n-butanolzethanolzwater (411:5 by volume) showsfl-bromoethylcephalosporin to be the major component. The activity ofthe products is determined by placing the paperchromatogram in contactwith S. aureus seeded plates.

The following cephalosporins are prepared by this procedure utilizingthe appropriate acid chloride in place of fi-bromopropionyl chloride.For convenience only the R group is listed. In each instance, thecorresponding acyl derivatives of Cephalosporin C and Cephalosporin Care also produced.

a- (p-Iodophenyl -fi-hydroxyethyl a-(p-Propylphenyl)-/3-hydroxyethylw-Carbobenzoxyaminopentyl fi-Carb obenzoxyaminoethyl'y-Carbobenzoxyaminopropyl 'y-Carb obenzoxyaminobutyl*y-Carbobenzoxyamino-[B-methyipropyl'y-Carbobenzoxyarnino-n-pentylcarbobenzoxy-aminobuty-lw-Carbobenzoxyaminohexyl ,B-Ca-rb obenzoxyamino-n-pentylfl-Carbobenzoxyamino-a-methylethylB-Carbobenzoxyamino-fi-ethylbutylcarbobenzoxyamino-n-heptylw-Carbobenzoxyaminoheptyl e-Carbobobenzoxyamino-y,y-dirnethylhexylw-Carbobenzoxyaminooctyl B-Carbobenzoxyamino-otphenylethylB-Carbobenzoxyamino-,B-phenylethyl Allylmercaptomethyl BromomethylChloromethyl ct-Chloroethyl p-Ohloroethyl a-Bromopropylot-Bromoisopropyl B-Bromopropyl a-Bromobutyl B-Bromobutyl fi-Chlorobutylu-Iodobutyl uo-Tolyl -,8-hydroxyethyl 0t- (m-Tolyl) 8-hydroxyethyl0cp-Tolyl) -,B-hydroxyethyl a- (o-Ethy-lphenyl -B-hydroxyethyl cc-(m-Ethylphenyl) fi hydroxyethyl up-Ethylphenyl) -fl-hydroxyethyl ec-(p-n-Butylphenyl) -B-hydr oxyethyl ocp-Is obutylphenyl -{3-hydroxyethyloz- (m-Bromophenyl) -fi-*hydroxyethyl a- (p-Bromophenyl)-fi-hydroxyethyl OL- (m-Chlorophenyl -[3-hydroxyethyl a- (p-Chlorophenyl-fit-hydroxyethyl vc- (p-Methoxyphenyl) -B-hydr0xyethyl a-(o-Nitrophenyl) -,B-hydroxyethyl a- (p-Nitrophenyl s-hydroxyethylOto-Butylphenyl -fi-hydroxyethyl O6- m-Methoxyphenyl) -fi-hy droxyethylao-Chloropheny-l fi hydroxyethyl a- (p-Fluorophenyl) -fl-hydroxyethyl a-(rn-trifluoromethylphenyl) 3- hydroxyethyl OL- o-Hydroxyphenyl)-/3-hydroxyethyl t-. (m-Hydroxyphenyl) -,8-hydroxyethylOtp-Hydroxyphenyl) e hydroxyethyl a- (p-Ethoxyphenyl) -/3-hydroxy ethylu-( o-Butoxyphenyl) -,3-hy droxyethyl a- (rn-Propoxyphenyl) -,B-hydroxyethyl u-Bromoamyl a-Chloroamyl fl-Bromoamyl a-Bromohexyla-Chlorohexyl B-Bromohexyl {3-Ohlorohexyl a-Bromoisoamyl wChloroisoamylIodomethyl a-Iodoethyl B-Iodo ethyl u-Allylmercaptoethyl u-Allylmercaptonapropyl ot-Allylmercapto-n-butyl a-Allylmercapto-n-pentyla-Allylmercapto-n-hexyl a-Allylmercapto-'y-rnethylbutyla-Allylmercapto-fi-methylpropyl Cis-styrylmercaptomethyl Transstyrylmercaptomethyl Pentyl Z-pentenyl Heptyl Phenylmercaptomethylp-Hydroxy-benzyl Example II POTASSIUM AMINOCEPHALOSPORIN A neutralaqueous solution of potassium cyanate (0.2 M) is added to a solution of7-aminocephalosporanic acid prepared as described in Procedure A, from 2g. of Cephalosporin C. Equimol-ar quantities of reactants are used basedon the Cephalosporin C used. The reaction mixture is stirred for onehour then adjusted to pH 7.0 with dilute potassium hydroxide and freezedried. The residue consists predominantly of the potassium salt of thecarbamyl derivative of 7-aminocephalosporanic acid, referred to hereinas arninocep-halosporin potassium salt. Smaller quantities of thecarbarnyl derivatives of Cephalosporin C and Cephalosporin C are alsoformed.

Alternatively, the products are isolated by adding the reaction mixturedropwise to a large excess of isopropyl alcohol. After chilling for 2hours, the products are recovered by filtration.

Example 111 A solution of 7-aminocephalosporanic'acid, preparedaccording to Procedure A above from 2 grams of Cephi anilinopenicillin.

In like manner the following N-aminocephalosporins are produced from theappropriate isocyanate. In cases wherein precipitation does not occurreadily, the reaction mixture is concentrated in vacuo to about /3volume and a large volume of ether added. The product is recovered byfiltration or decantation. The following N-substitutedaminocephalosporins are thus prepared: (only the R side chains arelisted):

1 a-Phenylmercapto p-hydroxyethyl tx-Phenylmercapto-B-hydroxy-n-propyltx-Phenylmercapto-B-hydroxy-n-hutyl a-Phenylmercapto-B-hydroxyn-pentylEthylarnino 5 a-Phenylmercapto-B-hydroxy-n-hexyl m-Propylaminoa-Phenylmercapto-fl-hydroxy-n-heptyl n-Butylamino Naphthyla 0a-Phenyirnercapto-fl-hydroxy-fi-methylpe tyl t Butylamin om-Phenylmercapto-B-hydroxy-a-methylbutyl n-Amylamino Example V lanfnnoThe a-phenoxy-B-hydroxyalkanoic acids used as ref z i actants in thepreceding example are converted to their y me acyl derivatives byrefluxing with an excess of the den-Decylamino sired acid chloride forone 'half hour. The excess acid Methylamino chloride is then removed bydistillation Under reduced (o-NitrophcnyD-amino pressure. Thionylchloride is added (a slight excess), (m-Nitrophenynamino the mixturerefluxed for one hour then distilled in vacuo (p-NitrophenyDamino toremove the excess thionyl chloride. The residue is(p-Met-hoXyphenyDamino reacted with 7-aminocephalosporanic acid as inExample (p-Dimethylaminophenyl)amino 20 I to give the acyl derivativesof the products of the (p-Carbomethoxyphenyl)amino preceding example. Inthis manner, the formyl, acetyl, (p-Ethoxyphenyl)amino propionyl andbutyryl derivatives of the products of Ex- (p-Chlorophenyllarnino ampleIV are obtained.

Example IV 2 Example VI 5 The following cephalospo-rins are prepared astheir Utilizing the procedure of Example I, but replaemg potassium saltsby the procedure of'EXample I using the fl-bromopropionyl chloride bythe proper 0,0-disubstiappropriate acid chloride. For convenience, onlythe tuted chloro thionophosphate or 0,0-disubstituted chloro- R groupsare listed: phosphate, the following cephalosporins, together with thecorresponding N-acylated derivatives of Cephalosporin (R) C andCephalosporin C are prepared. The R-C=O a-Phenoxy-fi-hydroxyethyl groupis replaced by a-Pl1enoxy-fl-hydroXy-n-propyl O aPhenoxy-fl-hydroxy-n-butyl a-Phenoxy-fi-hydroxy-n-pentyl n5a-Phenoxy-B-hydroxy-n-hexyl RPO :gg ggi j gigi fijgi z em 1 Forconvenience only the R and X values are listed. i f The listing of twovalues for X for a given value of R h' g: 'g 1 indicates that both thephosphoryl and thionophosphoryl en y 0 5 Y1) y 40 analogs are prepared.

R1 X B1 X Methyl S, O p Chlorophenylq S, 0 Ethyl... S, O o-BromophenyL-S, O lsopl'opyL S o-Nitrophenyl S, O Phenyl S, O .m-Nitrophenyl Sn-Butyl S -p-Nitropheny1 S, O fi-Methylpropyl. S, O o-Methoxyphenyl..-S, O ,B-Methoxyethyl S, O p-Methoxyphenyl S, O B-Chloroethyh. S, Oo-Ethoxyphenyl. S o-Tolyl S, O p-Propoxyphenyl S, O m-Tolyl..- S BenzylS, O p-Tolyl S, 0 a-Methylbenzyl.- S, O m-EthylphenyL- S, O-p-t-Butylphenyl S, 0 p-ButoxyphenyL- S, O o-Ghlorobenzyl. S, Op-Chlorobenzyl S inddethoxybenzylh S, O o-BromobenzyL S, O p-NitrobenzylS, O m-Methylbenzy S p-n-Butylbenzyl- S, 0 O-Propoxybenzyl.- So-PropylphenyL- S, 0 p-Isopropylphenyl S Ethyhnercaptoethyl S, 0Butylmereaptomethyl S, O fl-Phenylmercagitdethyl- Sfl-PhenylmereaptobutyLr S, O -n-Butoxyethyl S B-Methoxyheptyl S, On-Propoxyamyl S Methyhnereaptoheptvl S, O n-Propylmereaptoamyl S SPhenQ-n-heXyI" S, O Z-Phenoxy-n-butyl S, O w-Ethoxy-n-hexyl..- SB-Nitr0ethy1-. S,O w-Nitro-n-buty1 S, 0 w-Nitro-n-hexyl Sw-Nitro-n-heptyL S, O u-Ch10ro-n-heptyl S, O ,S-Bmmopropyl. Sm-Nitrobenzyl S o-Nitrobenzyl S, O p-t-Bntylbenzyl. S, O

S, O m-PhenylbutyL S S n-Pentyl S s s, o s S, 0 s s s s, 0 s, 0 s, 0 s sS, 0 s, o s s s, 0 s s, 0 s s s, 0 s

the condensation of the acid chloride of methylene-bisthioglycolic acidwith 7-arninocephalospor-anic acid.

Similarly additional related cephalosporins wherein the R group is o (lM- Rz-SRaS-R2 are prepared employing suitable acid chlorides. Only the Rand R values are listed, for convenience; M is hydroxyl.

Utilization of the monoamides, the monocarboxyinethyl andmonocarboxyethylidene substituted amides, and the monoesters of themonoacid chlorides corresponding to the R groups of the cephalosporinslisted above, produces the value of M is amide,carboxyrnethylimido,carboxye-thylideneimido and alkoxy containing 1 to 4 carbon atoms.

Example VIII The procedure of Example I is repeated usingwcarbobenzoxyarninocaproic acid chloride as the acylating agent to givew-carbobenzoxyaminopentylcephalosporin potassium salt.

The following compounds are prepared in like manner from the proper acidchloride (only the R groups are reported) B-Carbobenzoxyarninoethyl'y-Carbobenzoxyaminobutyl -Carbobenzoxyamino-n-pentylw-Oarbobenzoxyaminohexyl ,8-Carbobenzoxy-amino-u-rnethylethyl6-Carbobenzoxy-amino-mheptyl e-Carbobenzoxyamino-y, -dimethylhexylS-Carbobenzoxyamino-a-phenylethyl -Carbobenzoxyarninopropyl-Carbobenzoxyarnino-fi-methylpropyl fi-carbobenzoxyaminobutylfi-Carbobenzoxyamino-n-pentyl B-Carbobenzoxyamino-B-ethylbutylw-carbobenzoxyaminoheptyl w-C-arbobenzoxyaminooctylfi-Carbobenzoxy-amino-,B-phenylethyl Exam pile IX A solution ofw-carbobenzoxyaminopentylcephalosporin potassium salt is acidified to pH2.5 with aqueous phosphoric acid and extracted with two one-half volumesof n-butanol. The n-butanol extracts are combined and washed with water.

The n-butanol solution is then added to a hydrogenation flask containingan equal volume of Water, 0.5 g. of palladium black and the systemstirred under hydrogen until evolution of carbon dioxide no longeroccurs. The reaction mixture is then filtered and the filter cake Washedwith n-butanol and Water. To the combined filtrate and washings there isadded one-half volume of water-n-butanol (2:1), the mixture thoroughlyagitated and the layers separated. The aqueous layer is then freezedried to give w-aminopentylpenicillin.

Hydrogenolysis of the remaining products of Example VII produces thecorresponding aminoalkylcephalosporins having the R group.

fl-Aminoethyl 'y-Arninopropyl- 'y-Aminobutyl *y-Amino-B-methylprop-yl'y-Amino-n-pentyl 6-Amin0butyl w-Aminohexyl fl-Amino-n-pentylfl-Amino-u-methylethyl fl-Arnino-fl-ethylbutyl fi-Amino-n-heptylw-Aminoheptyl e-Amino-wy-dimethylhexyl w-Aminooctylfi-Amino-u-phenylethyl fl-Arnino-fl-phenylethyl Example X TheCephalosporin C products corresponding to the above cephalosporins areproduced by substituting the precursor of Cephalosporin C for7-amino-cephalosporanic acid in the preceding examples.

13 Example XI The products of the preceding examples are converted totheir free acid forms by neutralization of the aqueous solutions oftheir potassium salts with 6 N hydrochloric acid to pH 2.2. The acidsare recovered by extraction into methylisobutylketone followed byevaporation of the solvent.

Example XII The free acids of Example VIII are transformed to theirsodium, calcium, ammonium, procaine, N,N-dibenzylethylenediamine.dibenzylamine, l-ephenamine, N- benzyl-B-pheuethylamine, andbenzhydrylamine salts by reaction of aqueous solutions thereof with oneequivalent of the appropriate base. The salts are recovered by freezedrying.

What is claimed is:

1. The compound having the formula wherein R' is acetoxy and R ischloro-, brornoand iodosubstituted alkyl having from 1 up to 6 carbonatoms.

2. The compound having the formula wherein R is acetoxy and R isa-phenyl-fi-hydroxyalkyl having up to carbon atoms in the alkyl group.

3. The compound having the formula wherein R is acetoxy and R isa-phenoxy-fi-hydroxyalkyl having from 2 up to 5 carbon atoms in thealkyl group.

4. The compound having the formula wherein R is acetoxy and R is:x-phenoXy-fl-acetoxyalkyl having from 2 up to 5 carbon atoms in thealkyl group.

5. The compound having the formula wherein R is acetoxy and R isaminoalkyl having from 1 up to 8 carbon atoms.

6. The compound having the formula wherein R is acetoxy and R isa-allylmercaptoalkyl having up to 6 carbon atoms in the alkyl group.

7. The compound having the formula wherein R is acetoxy and R is alkylsubstituted amino having from 1 up to 10 carbon atoms.

8. The compound having the formula References Cited by the ExaminerUNITED STATES PATENTS 6/60 Doyle et a1. 260-239.1

OTHER REFERENCES Burger: Medicinal Chemistry, pages 36-48 (1969).Journal American Medical Assoc., page 466, May 24, 1958.

NICHOLAS S. RIZZO, Primary Examiner. I. MARCUS, WALTER A. MODANCE,Examiners.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,175,916 March 16, 1965 Gilbert M. Shull et al,

It is hereby certified that error appears in theabove numbered patentreqiiring correction and that the said Letters Patent should read ascorrectedbelow.

Column 1, line 65 for "5-carboxyv1leryl" read 6-carboxyvaleryl columnll, .Example VII, in the table, heading to the first column, for "R readR Signed and sealed this 24th day of August 1965,.

SEAL) Ltest:

RNEST w. SWIDER EDWARD J. BRENNER lusting Officer Commissioner ofPatents

3. THE COMPOUND HAVING THE FORMULA
 5. THE COMPOUND HAVING THE FORMULA13. AMINOCEPHALOSPORIN.
 14. O,O-DIETHYLTHIONOPHOSPHORYLCEPHALOSPORIN.